Persistent benzodiazepine (BZD) use is commonly associated with the development of tolerance to its sedative and anti-convulsant actions. Despite decades of research, the mechanisms underlying these phenomena are poorly understood. The classical BZD, diazepam (DZP), potentiates the actions of the inhibitory neurotransmitter GABA at GABA type A receptors (GABAARs); namely those containing ?1/?2/?3/?5 subunits and a ?2 subunit. Importantly, the ?2 subunit is critical for the majority of DZP-sensitive GABAAR complexes. The inhibitory synaptic scaffolding protein gephyrin acts to tether GABAARs at synapses opposed to axon terminals releasing GABA. Phosphorylation of gephyrin at a serine 270 residue can promote its degradation and declustering, reducing scaffolding of GABAARs. Dynamic changes in gephyrin ultimately affect the rate of synaptic GABAAR turnover and receptor movement into the extrasynaptic space where internalization can occur. Importantly, our lab has previously shown 24 hr BZD exposure enhances lysosomal-mediated degradation of ?2-GABAARs in cultured neurons. We hypothesize that DZP exposure reduces gephyrin synaptic scaffolding, promoting internalization and lysosomal targeting of ?2-GABAARs. This hypothesis will be tested by the following aims: 1) Determine the effect of DZP treatment on ?2-containing GABAAR surface regulation; and 2) Evaluate mechanisms regulating gephyrin scaffolding function following DZP exposure. In aim 1, we will determine the effect of DZP on ?2 surface and total levels in primary rat cortical neurons and in mouse cortical tissue. These studies will also investigate the role of ?2 subunit ubiquitination and lysosomal targeting in the loss of DZP sensitivity. A lysine ubiquitin-null ?2 mutant subunit will be incorporated into an adeno-associated virus (AAV) to be used for in-vitro and in-vivo transfection. Aim 2 will assess gephyrin phosphorylation and total levels in response to DZP. Live-imaging techniques will assess gephyrin scaffolding of GABAARs at synapses after DZP exposure. To compare gephyrin-synaptic GABAAR interactions in-vivo, we will take a quantitative proteomic approach to compare ?2-GABAAR interacting proteins between DZP and vehicle control mice. These studies will ultimately identify broad spectrum changes in synaptic GABAAR protein association after DZP. Pilot studies using our ?2 coimmunoprecipitation-mass spectrometry method have identified gephyrin and all anticipated GABAAR subunits that contribute to DZP- sensitive GABAARs, along with 575 matching proteins from a similar published proteomic study using GFP- tagged ?2-GABAARs. These proteomic analyses will also include screening of gephyrin phospho-peptide levels in-vivo. Together, these studies will provide fundamental information about the role of ?2-GABAAR trafficking and altered protein interactions in response to DZP treatment. During the completion of this work, the candidate will gain experience performing in-vitro and in-vivo experimental techniques including biochemistry, molecular biology, proteomics, and multiple imaging applications.